P53 binds preferentially to non-B DNA structures formed by the pyrimidine-rich strands of GaA·TTC trinucleotide repeats associated with Friedreich’s ataxia

Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich’s ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich’s ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs’ non-B DNA structures

p53 Specifically Binds Triplex DNA In Vitro and in Cells

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed

Verification of candidate p53 target genes.

<p><b>(A)</b> RT-qPCR analysis of candidate p53 target genes and BAX, p21 and p53 mRNA levels in i) H1299 cells transfected by pCDNAp53 for 48 hours (left graph); ii) MCF7 cells with downregulation of p53 by siRNA over control siRNA for 48 hours (right graph). <b>(B)</b> RT-qPCR analysis of candidate p53 target genes and <i>BAX</i>, <i>p21</i> mRNA levels in MCF7 cells after nutlin-3 or actinomycin D 12 hours treatment. Gene values were normalized to GAPDH. The values are the average of three independent experiments. <b>(C)</b> p53 mediated up-regulation of NAT10 on protein level and activation of BAX and CDKN1A was analyzed in Hwtp53 cells (24 hours induction) vs H1299 without p53 expression. Western blots presenting protein levels of p53, NAT10, CDKN1A and BAX. Actin was used as loading control. <b>(D)</b> Chromatin immunoprecipitation showing p53 binding to <i>MCC</i> and <i>NAT10</i> promoters which contain a TAT triplex motif. DNA fragments from MCF7 cells without and with nutlin-3/doxorubicin 4 hours treatment were immunoprecipitated using DO1 antibody against p53 (lane 4,7,10), negative control ChIP with IgG (lanes 3,6,9), positive input control (1/15 input for ChIP, lanes 2,5 and 8).</p

Influence of T.A.<i>T</i> triplex forming sequence on p53-driven activation of CON containing reporter vector in scDNA and lin DNA.

<p><b>(A)</b> Scheme of reporter plasmid constructs used in luciferase reporter assay and non-B DNAs formation under supercoiled stress (CF- cruciform, TAT-triplex). <b>(B-C)</b> H1299 cells were transiently transfected with plasmids expressing the p53 (pCDNA3.1-p53) or pCDNA3.1 vector alone (CMV) together with reporter: the supercoiled or linear reporter plasmids (BSK, P1, P20, P50, B50) expressing the firefly luciferase gene and a reference plasmid with the renilla gene under control of the SV40 promoter. Luciferase activity was analyzed 16 hours after transfection and signal was normalized on renilla signal. Transfections were carried out in triplicates at least at three independent times and standard deviations are indicated. <b>(B)</b> p53 activation of supercoiled reporters. Luciferase activity was normalized on control with vector alone. Only B50 and P50 reporters were able to form triplexes. p53 activation of linear reporter as described above, none of used reporters was able to form triplexes. <b>(C)</b> p53 activation of supercoiled reporter plasmids in H1299-wtp53 cells (Tet-on promoter). Luciferase signal after p53 induction was normalized on control without p53 induction. Only B50 and P50 reporters were able to form triplexes. <b>(D)</b> Interaction of full length p53 with CON (P1) and triplex T.A.<i>T</i> (B50) in scDNA plasmids by ChIP <i>in vivo</i>. Plasmids BA50 or PGM1 (2 μg) were transfected into H1299 cells together with vector pCDNA3.1-wtp53 (0.1 μg). ChIP was performed with CM1 antibody. Results of PCR analyses of immunoprecipitated DNA were detected on a 1.5% agarose gel in 1× TAE buffer. PCR samples on the gel are: marker (lane 1), plasmid PGM1 (P1, lane 2) and BA50 (lane 6); 1/20 of DNA input (lanes 5 and 9 marked as IN); IP with IgG (negative control) (lanes 4 and 8); IP with CM1 Ab (lanes 3 and 7).</p

Binding of p53 to supercoiled DNA bearing homoadenine-homothymine triplex forming sequences.

<p><b>(A)</b> Scheme of intramolecular T.A.<i>T</i> triplex in scDNA. AFM image of sc pA69 plasmid adsorbed on mica surface in the presence of 2 mM MgCl₂ and complex of pA69 with p53. <b>(B)</b> Comparison of p53 binding to scDNA with and without triplex forming sequence (dA)₅₀.(dT)₅₀ by EMSA. Binding of p53 protein to pBSK, pPGM1, pBA50 and pPA50 detected by EMSA in agarose gel. p53 protein was bound to scDNA (pBSK, 200 ng, lanes 1–5), scDNA with CON (scPGM1, 200 ng, 6–10), scDNA with (dA)₅₀.(dT)₅₀ (scBA50, 200 ng, 11–15) and scDNA with both CON and (dA)₅₀.(dT)₅₀ (scPA50, 200 ng, 16–20) in p53/DNA molar ratios 1–3 at 4°C, EMSA was performed at 4°C. Graph represents the dependence of percents of bound DNA on the amount of p53 proteins calculated from three experiments. <b>(C)</b> Interaction of p53 with scDNA (BSK, PGM1, BA50 and PA50) in presence of pBSK/SmaI (linear competitor, lin) by immunoprecipitation on MBG. Agarose gel electrophoresis of DNA recovered from MBG after incubation of DO1-wtp53-DNA complex at the beads to 50, 100, 300 or 600 mM KCl for 30 min at 10°C followed by the SDS treatment. DNA inputs of scDNA BSK (lane 2), PGM1 (lane 3), BA50 (lane 4), PA50 (lane 5), linBSK (lane 1). Arrows indicate precipitated supercoiled (sc), open circular (oc), linear (lin) and supercoiled dimers (dimer sc). Mean values of bound DNA from three independent experiments were plotted in the graph. Graph represents the dependence of percents of bound DNA on the concentration of KCl in washing buffer calculated from three experiments. <b>(D)</b> Competition assay of p53 binding to CON and non-B-DNA structures in scDNA plasmids. First, full length p53 (60 ng) was incubated with 200 ng PGM1/<i>Pvu</i>II fragments (short fragment with CON sequence (CON, 474 bp) and long fragment as linear nonspecific competitor (NON, 2513 bp) for 20 min on ice to form p53-CON complexes. Subsequently, 200 or 300 ng of different scDNA plasmid competitors were added and incubation was prolonged to 40 min. Plasmids forming triplex T.A.<i>T</i> were marked by TAT, plasmids forming cruciform by X. Graph represents the dependence of percents of bound DNA on the amount of used competitor scDNAs calculated from three experiments.</p

Full length p53 binds strongly to T.A.<i>T</i> triplex DNA.

<p><b>(A)</b> Full length p53 was incubated with 1 pmol of ³²P-labeled 50-mer oligonucleotides: nonspecific dsDNA (NON, lanes 1–5), p53 specific dsDNA with CON (CON, lanes 6–10) and (dT)₅₀.(dA)₅₀.(dT)₅₀ triplex (T.A.<i>T</i> triplex, lanes 11–15) in presence of 50 ng pBSK/<i>Sma</i>I. Molar ratios of p53 tetramer/DNA ranged between 0.1 and 0.75. The samples were loaded onto 5% 0.5 × TBM (2 mM MgCl₂) polyacrylamide gel and electrophoresis was performed for 0.45 h. <b>(B)</b> Full length p53 was incubated with 1 pmol of ³²P-labeled (dT)₅₀.(dA)₅₀.(dT)₅₀ triplex (T.A.<i>T</i> triplex, lanes 1–5), CTG hairpin (lanes 6–10) and TA hairpin (lanes 11–15) oligonucleotides in presence of 50 ng pBSK/<i>Sma</i>I. Molar ratios of p53 tetramer/DNA ranged between 0.2 and 1.2. The samples were loaded onto 5% 0.5 × TBM (2 mM MgCl₂) polyacrylamide gel and electrophoresis was performed for 0.45 h. <b>(C)</b> p53 binding to biotinylated oligonucleotides by ELISA. p53 binding curves for the TAT, CON and CTG oligonucleotides are shown, and the dissociation constants (Kd) are indicated.</p

Binding of p53CD and C-terminal p53 fragments to T.A.<i>T</i> triplex.

<p><b>(A)</b> p53 Core domain (p53CD, aa 94–312) and full length p53 were bound to CON, (lanes 1–7) and triplex (TAT, lanes 8–14) in p53 tetramer/DNA molar ratios 0.7–10 in presence of 10 ng competitor DNA. Graph of p53CD (aa 94–312) binding to biotinylated oligonucleotides by ELISA. p53CD binding curves for the TAT, CON and A oligonucleotides are shown, and the dissociation constants (Kd) are indicated. <b>(B)</b> C-terminal part of p53 (p53CT, aa 320–393) was incubated with (dT)₅₀ (T, lanes 1–5), triplex (dT)₅₀.(dA)₅₀.(dT)₅₀ (TAT, lanes 6–10) and CON (lanes 11–15) in p53CT tetramer/DNA molar ratios 0.4–3.6. Graph p53CT (aa 320–393) binding to biotinylated oligonucleotides by ELISA. p53CT binding curves for the TAT, CON and A oligonucleotides are shown, and the dissociation constants (Kd) are indicated. <b>(C)</b> C-terminal part of p53 (p53T, aa 363–393) was incubated with (dA)₅₀ (A, lanes 1–5), triplex (dT)₅₀.(dA)₅₀.(dT)₅₀ (TAT, lanes 6–10) and double-stranded TA (lanes 11–15) in p53CT tetramer/DNA molar ratios 0.8–8.4. Graph of p53T (aa 363–393) binding to biotinylated oligonucleotides by ELISA. p53T binding curves for the TAT, CON and A oligonucleotides are shown, and the dissociation constants (Kd) are indicated <b>(D)</b> Scheme showing p53 domains and p53 protein constructs used in this work. <b>(E)</b> Relative binding properties of p53 protein constructs to TAT triplex and CON oligonucleotides.</p